CN107236011B - Nucleoside compound or salt thereof, nucleic acid and application thereof - Google Patents
Nucleoside compound or salt thereof, nucleic acid and application thereof Download PDFInfo
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- CN107236011B CN107236011B CN201710456982.9A CN201710456982A CN107236011B CN 107236011 B CN107236011 B CN 107236011B CN 201710456982 A CN201710456982 A CN 201710456982A CN 107236011 B CN107236011 B CN 107236011B
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 64
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 56
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 56
- 150000003839 salts Chemical class 0.000 title claims abstract description 47
- -1 Nucleoside compound Chemical class 0.000 title abstract description 56
- 239000002777 nucleoside Substances 0.000 title abstract description 15
- 108091023037 Aptamer Proteins 0.000 claims abstract description 48
- 108091027757 Deoxyribozyme Proteins 0.000 claims abstract description 36
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 25
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 claims abstract description 18
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 claims abstract description 18
- 102000044890 human EPO Human genes 0.000 claims abstract description 18
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 13
- 230000007017 scission Effects 0.000 claims abstract description 13
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 claims abstract description 12
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 claims description 59
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 claims description 26
- 125000003729 nucleotide group Chemical group 0.000 claims description 21
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 16
- 230000004048 modification Effects 0.000 claims description 10
- 238000012986 modification Methods 0.000 claims description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 229910021645 metal ion Inorganic materials 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 230000003511 endothelial effect Effects 0.000 claims description 4
- 125000004437 phosphorous atom Chemical group 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 4
- 230000002792 vascular Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 claims description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 128
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 117
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 72
- 238000006243 chemical reaction Methods 0.000 description 61
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 48
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 33
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 24
- 230000015572 biosynthetic process Effects 0.000 description 23
- 238000003786 synthesis reaction Methods 0.000 description 23
- 238000004809 thin layer chromatography Methods 0.000 description 23
- 239000000047 product Substances 0.000 description 22
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 21
- 238000005160 1H NMR spectroscopy Methods 0.000 description 21
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 20
- 238000004440 column chromatography Methods 0.000 description 20
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 18
- 229910052757 nitrogen Inorganic materials 0.000 description 15
- 238000000034 method Methods 0.000 description 14
- 108090000994 Catalytic RNA Proteins 0.000 description 12
- 102000053642 Catalytic RNA Human genes 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 12
- 108091092562 ribozyme Proteins 0.000 description 12
- 239000002904 solvent Substances 0.000 description 12
- JBWYRBLDOOOJEU-UHFFFAOYSA-N 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene Chemical compound C1=CC(OC)=CC=C1C(Cl)(C=1C=CC(OC)=CC=1)C1=CC=CC=C1 JBWYRBLDOOOJEU-UHFFFAOYSA-N 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 11
- 125000003258 trimethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])[*:1] 0.000 description 11
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 10
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 description 10
- 125000000623 heterocyclic group Chemical group 0.000 description 10
- 125000002883 imidazolyl group Chemical group 0.000 description 10
- 239000000178 monomer Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 230000003197 catalytic effect Effects 0.000 description 9
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 8
- UOXMJKDGJMTPSI-UHFFFAOYSA-N n-ethenyl-2,2,2-trifluoroacetamide Chemical group FC(F)(F)C(=O)NC=C UOXMJKDGJMTPSI-UHFFFAOYSA-N 0.000 description 8
- CIXHNHBHWVDBGQ-UHFFFAOYSA-N n-propan-2-ylpropan-2-amine;2h-tetrazole Chemical compound C1=NN=NN1.CC(C)NC(C)C CIXHNHBHWVDBGQ-UHFFFAOYSA-N 0.000 description 8
- 239000007858 starting material Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 7
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 description 7
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- CBHOOMGKXCMKIR-UHFFFAOYSA-N azane;methanol Chemical compound N.OC CBHOOMGKXCMKIR-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 150000003833 nucleoside derivatives Chemical class 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- UFPBWCBCUAYIIW-UHFFFAOYSA-N 2,2,2-trifluoro-n-prop-2-enylacetamide Chemical group FC(F)(F)C(=O)NCC=C UFPBWCBCUAYIIW-UHFFFAOYSA-N 0.000 description 5
- 108010065108 RNA-cleaving DNA 10-23 Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- STSCVKRWJPWALQ-UHFFFAOYSA-N TRIFLUOROACETIC ACID ETHYL ESTER Chemical compound CCOC(=O)C(F)(F)F STSCVKRWJPWALQ-UHFFFAOYSA-N 0.000 description 5
- 125000003368 amide group Chemical group 0.000 description 5
- MDFFNEOEWAXZRQ-UHFFFAOYSA-N aminyl Chemical group [NH2] MDFFNEOEWAXZRQ-UHFFFAOYSA-N 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 150000008300 phosphoramidites Chemical class 0.000 description 5
- 230000000865 phosphorylative effect Effects 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 235000017557 sodium bicarbonate Nutrition 0.000 description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- HBENZIXOGRCSQN-VQWWACLZSA-N (1S,2S,6R,14R,15R,16R)-5-(cyclopropylmethyl)-16-[(2S)-2-hydroxy-3,3-dimethylpentan-2-yl]-15-methoxy-13-oxa-5-azahexacyclo[13.2.2.12,8.01,6.02,14.012,20]icosa-8(20),9,11-trien-11-ol Chemical compound N1([C@@H]2CC=3C4=C(C(=CC=3)O)O[C@H]3[C@@]5(OC)CC[C@@]2([C@@]43CC1)C[C@@H]5[C@](C)(O)C(C)(C)CC)CC1CC1 HBENZIXOGRCSQN-VQWWACLZSA-N 0.000 description 4
- 238000004679 31P NMR spectroscopy Methods 0.000 description 4
- 125000000242 4-chlorobenzoyl group Chemical group ClC1=CC=C(C(=O)*)C=C1 0.000 description 4
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 125000004185 ester group Chemical group 0.000 description 4
- UCSVJZQSZZAKLD-UHFFFAOYSA-N ethyl azide Chemical compound CCN=[N+]=[N-] UCSVJZQSZZAKLD-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- AOHJOMMDDJHIJH-UHFFFAOYSA-N propylenediamine Chemical compound CC(N)CN AOHJOMMDDJHIJH-UHFFFAOYSA-N 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- SHAHPWSYJFYMRX-GDLCADMTSA-N (2S)-2-(4-{[(1R,2S)-2-hydroxycyclopentyl]methyl}phenyl)propanoic acid Chemical compound C1=CC([C@@H](C(O)=O)C)=CC=C1C[C@@H]1[C@@H](O)CCC1 SHAHPWSYJFYMRX-GDLCADMTSA-N 0.000 description 3
- VIMMECPCYZXUCI-MIMFYIINSA-N (4s,6r)-6-[(1e)-4,4-bis(4-fluorophenyl)-3-(1-methyltetrazol-5-yl)buta-1,3-dienyl]-4-hydroxyoxan-2-one Chemical compound CN1N=NN=C1C(\C=C\[C@@H]1OC(=O)C[C@@H](O)C1)=C(C=1C=CC(F)=CC=1)C1=CC=C(F)C=C1 VIMMECPCYZXUCI-MIMFYIINSA-N 0.000 description 3
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 3
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- YLEIFZAVNWDOBM-ZTNXSLBXSA-N ac1l9hc7 Chemical compound C([C@H]12)C[C@@H](C([C@@H](O)CC3)(C)C)[C@@]43C[C@@]14CC[C@@]1(C)[C@@]2(C)C[C@@H]2O[C@]3(O)[C@H](O)C(C)(C)O[C@@H]3[C@@H](C)[C@H]12 YLEIFZAVNWDOBM-ZTNXSLBXSA-N 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
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- 125000003118 aryl group Chemical group 0.000 description 3
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 3
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- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
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- RWWYLEGWBNMMLJ-YSOARWBDSA-N remdesivir Chemical compound NC1=NC=NN2C1=CC=C2[C@]1([C@@H]([C@@H]([C@H](O1)CO[P@](=O)(OC1=CC=CC=C1)N[C@H](C(=O)OCC(CC)CC)C)O)O)C#N RWWYLEGWBNMMLJ-YSOARWBDSA-N 0.000 description 3
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- KAFZOLYKKCWUBI-HPMAGDRPSA-N (2s)-2-[[(2s)-2-[[(2s)-1-[(2s)-3-amino-2-[[(2s)-2-[[(2s)-2-(3-cyclohexylpropanoylamino)-4-methylpentanoyl]amino]-5-methylhexanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]butanediamide Chemical compound N([C@@H](CC(C)C)C(=O)N[C@@H](CCC(C)C)C(=O)N[C@@H](CN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(N)=O)C(N)=O)C(=O)CCC1CCCCC1 KAFZOLYKKCWUBI-HPMAGDRPSA-N 0.000 description 2
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- 125000003785 benzimidazolyl group Chemical group N1=C(NC2=C1C=CC=C2)* 0.000 description 2
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- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
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- 239000001177 diphosphate Substances 0.000 description 2
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
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- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/505—Erythropoietin [EPO]
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- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
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- Biomedical Technology (AREA)
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- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
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- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of biological medicine, and relates to a nucleoside compound or salt thereof, nucleic acid and application thereof. Specifically, the invention relates to nucleoside compounds shown as a formula I or salts thereof; on the basis, the invention also relates to nucleic acids. The nucleic acid of the present invention can be used as a deoxyribozyme that catalyzes the cleavage of mRNA of vascular endothelial growth factor receptor 2 or as an aptamer having an affinity for human erythropoietin.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a nucleoside compound or a salt thereof, a nucleotide compound or a salt thereof, nucleic acid, and application of the nucleoside compound or the salt thereof, the nucleotide compound or the salt thereof and the nucleic acid.
Background
Since the early eighties, the discovery of naturally occurring ribozymes by Cech has led us to re-recognize the function of nucleic acids. In addition to being a carrier of genetic information, nucleic acids (e.g., RNA fragments) are also present in cells and are actively involved in regulating certain processes of life. In addition, in vitro selection evolution enrichment technology (in vitro selection) is utilized to obtain nucleic acids with novel functions, wherein the nucleic acids mainly comprise two major classes, one class is ribozymes or deoxyribozymes for catalyzing phosphodiester bond linkage or cleavage of nucleic acids, and the other class is aptamers.
Ribozymes and deoxyribozymes are mainly used in the field of gene therapy. For example, recognition fragments (recognition domains) of ribozymes and deoxyribozymes are designed for fragments (target sequences) of pathogenic mRNA, and specific recognition and catalytic cleavage occur through the complementarity of the recognition fragments and the target sequences, so as to destroy the pathogenic mRNA and interrupt the subsequent translation of the pathogenic mRNA into pathogenic protein.
An aptamer is a nucleic acid molecule that has been screened under conditions for affinity for a particular target, including metal ions associated with environmental contamination (e.g., Hg)2+，Pb2+) Signal molecules associated with the occurrence of diseases, abnormally expressed cells (tumor cells, etc.), diseased tissues, and foreign viruses and bacteria. Therefore, the aptamer with high specificity and high affinity can be used as a probe for detecting metal ions or disease signal molecules and can also be used as a therapeutic drug for inhibiting pathogenic protein molecules, viruses, bacteria, cells and tissues.
Based on the huge application potential of ribozymes, deoxyribozymes and aptamers, finding out ribozymes and deoxyribozymes with stronger catalytic ability and aptamers with higher affinity and specificity is a great research hotspot at present. There are two main discovery strategies available: the first method is that the screened ribozymes, deoxyribozymes and aptamers with application values are structurally modified by modifying basic groups to optimize functions, improve the catalytic capability of the ribozymes or deoxyribozymes, or improve the affinity and specificity of the aptamers to a target; the second is to introduce modified bases or modified nucleotides into a nucleic acid sequence library, and screen out nucleic acids with stronger functions from the head by using methods such as in vitro screening evolution enrichment technology (in vitro selection) and the like, wherein the nucleic acids comprise ribozymes or deoxyribozymes with higher catalytic efficiency, ribozymes or deoxyribozymes for catalyzing novel reactions, and aptamers with higher affinity and specificity to targets.
The modification of the basic group is mainly to introduce a functional group to change the higher structure and the biological activity of the nucleic acid, so as to screen out the functional nucleic acid with better performance. There are two main methods for base modification: one is to introduce carboxyl, amino, hydroxyl, sulfydryl or ester group by taking alkenyl or alkynyl as a connecting arm through palladium-catalyzed coupling reaction by means of halogen atoms, and then introduce a functional group by means of amide bond formation or ether bond formation and the like; the other is to modify the base by de novo synthesis, introduce carboxyl groups on the pyrimidine, and then introduce functional groups via the carboxyl groups.
In vitro selection evolution enrichment technology (in vitro selection) is based on the use of target nucleic acids in a nucleic acid sample containing about 1014-1016The screening conditions and standards are set in the library of the nucleic acid sequences, under certain screening conditions, the nucleic acid sequences form various high-level structures, no matter cracked or connected nucleic acid is used as the screening standard, or various molecules with high affinity and high specificity are used as the screening standard, and the screened nucleic acid sequences with activity are subjected to multiple rounds of amplification and re-screening to obtain the target nucleic acid with highest activity. Typical deoxyribozymes include 10-23 deoxyribozyme and 8-17 deoxyribozyme, and the aptamers include thrombin aptamers, small-molecule ATP aptamers, tetracycline aptamers, cocaine aptamers, nicotine aptamers, and the like.
There is a need to obtain ribozymes or deoxyribozymes with stronger catalytic ability and aptamers with stronger affinity.
Disclosure of Invention
The invention provides a nucleoside compound or a salt thereof. The invention also provides a nucleotide compound or a salt thereof. On the basis, the invention also provides a nucleic acid which can be used as a deoxyribozyme for catalyzing the mRNA cleavage of the vascular endothelial growth factor receptor 2 or an aptamer with an affinity action with human erythropoietin, and can be used for inhibiting the growth of vascular endothelium or detecting human erythropoietin.
The invention relates in a first aspect to a compound of formula I or a salt thereof,
wherein,
r is selected from hydroxyl, sulfydryl, substituted or unsubstituted amino and C1-8Alkoxy, benzyloxy, -O (CH)2)mR’、-S(CH2)mR’、-NH(CH2)mR’、-N[(CH2)mR’]2And- (CH)2)mR'; it is composed ofWherein R' is selected from the group consisting of substituted or unsubstituted hydroxyl, mercapto, substituted or unsubstituted amino, substituted or unsubstituted guanidino, C5-20Aryl radical, C3-20Heterocyclyl, ester, amide, and substituted imidazolyl, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R1selected from-H, substituted or unsubstituted hydroxyl, halogen, substituted or unsubstituted amino, azido, C1-8Alkoxy, O-allyl, C1-8Alkoxy ethoxy, C1-8Alkylamino radical and C3-15A cycloalkylamino group;
R2selected from the group consisting of-H, 4' -dimethoxytriphenylmethyl, 4-methoxytriphenylmethyl, monophosphate and salts thereof, diphosphate and salts thereof, and triphosphate and salts thereof;
R3is-H or PN (iPr)2OCH2CH2CN；
n is 1, 2, 3, 4, 5, 6, 7 or 8;
wherein the substituted hydroxy group is a hydroxy group substituted with: acetyl, benzoyl, p-methylbenzoyl, p-chlorobenzoyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; substituted amino is amino that is mono-or polysubstituted with substituents selected from the group consisting of: trifluoroacetyl, benzoyl, p-methylbenzoyl, p-chlorobenzoyl, acetyl and phthaloyl; substituted imidazolyl is imidazolyl that is mono-or polysubstituted with substituents selected from the group consisting of: t-butoxycarbonyl, triphenylmethyl, 4-methoxytriphenylmethyl and 4, 4-dimethoxytriphenylmethyl; a substituted guanidino group is a guanidino group that is mono-or poly-substituted with a substituent selected from the group consisting of: t-butyloxycarbonyl acyl and cyanoethoxycarbonyl acyl.
In one embodiment of the first aspect of the present invention, R is selected from the group consisting of hydroxy, mercapto, amino, -O (CH)2)mR’、-S(CH2)mR’、-NH(CH2)mR’、-N[(CH2)mR’]2And- (CH)2)mR'; wherein R' is selected from hydroxyl, sulfydryl, amino, guanidino and C5-20Aryl radical, C3-20Heterocyclyl, ester and amide groups, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In one embodiment of the first aspect of the present invention, R is-NH (CH)2)mR ', wherein R' is selected from amino, guanidino and imidazolyl, and m is 2 or 3.
In one embodiment of the first aspect of the present invention, R1Selected from-H, hydroxy, halogen, amino, azido, C1-8Alkoxy, O-allyl, C1-8Alkoxy ethoxy, C1-8Alkylamino radical and C3-15A cycloalkylamine group.
In one embodiment of the first aspect of the present invention, R1Selected from the group consisting of-H, hydroxy, fluoro, amino, azido, methoxy, ethoxy, propoxy, O-allyl, methoxyethoxy, ethoxyethoxy, propoxyethoxy, methylamino, ethylamino, propylamino, and cyclopropylamino.
In one embodiment of the first aspect of the present invention, R2Selected from the group consisting of-H, 4' -dimethoxytriphenylmethyl, 4-methoxytriphenylmethyl, monophosphate, diphosphate and triphosphate.
In one embodiment of the first aspect of the present invention, R2is-H.
In one embodiment of the first aspect of the present invention, R3is-H or PN (iPr)2OCH2CH2CN。
In one embodiment of the first aspect of the present invention, R3is-H.
In one embodiment of the first aspect of the present invention, n is 1, 2, 3, 4, 5 or 6.
In one embodiment of the first aspect of the present invention, n is 1 or 2.
In one embodiment of the first aspect of the present invention, R is selected from-NH (CH)2)mR'; wherein R' is selected from the group consisting of substituted amino, substituted guanidino and substituted imidazolyl, and m is 2 or 3.
In one embodiment of the first aspect of the present invention, R1is-H.
The inventionIn one embodiment of the first aspect, R2is-H or 4, 4' -dimethoxytriphenylmethyl.
In one embodiment of the first aspect of the present invention, R3is-H or PN (iPr)2OCH2CH2CN。
In one embodiment of the first aspect of the present invention, n is 1 or 2.
In one embodiment of the first aspect of the invention, it is selected from the following compounds and salts thereof:
5- (2-aminoethyl) -aminylmethylene-2' -deoxyuridine;
5- [ N- (2-trifluoroacetamidoethylene) amidomethylene ] -2' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamidoethylene) amidooxymethylene ] -2 ' -deoxyuridine;
5 '- (4, 4' -dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamidoethylene) amidomethylene ] -2 '-deoxyuridine-3' - (2-cyanoethylenoxy) -N, N-diisopropylamidophosphoramidite;
5- (3-aminopropylamidomethylene) -2' -deoxyuridine;
5- [ N- (3-trifluoroacetamidopropylene) amidoylmethylene ] -2' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) amidomethylene ] -2 ' -deoxyuridine;
5 '- (4, 4' -dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) amidomethylene ] -2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylamidophosphoramidite;
5- [ 2-aminoethyl ] aminyl ethylene-2' -deoxyuridine;
5- [ N- (2-trifluoroacetamido ethylene) aminyl ethylene ] -2' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamidoethylene) amidoethylene ] -2 ' -deoxyuridine;
5 '- (4, 4' -dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamidoethylene) amidoethylene ] -2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylamidophosphoramidite;
5- [ (3-aminopropyl) -aminoylethylene ] -2' -deoxyuridine;
5- [ N- (3-trifluoroacetamidopropylene) amidoethylenyl ] -2' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylene) amidoethylene ] -2 ' -deoxyuridine;
5 '- (4, 4' -dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) -amidoethylenyl ] -2 '-deoxyuridine-3' - (2-cyanoethylenyl-O-) -N, N-diisopropylamidophosphoramidite;
5- [ 2-imidazolyl-4- ] ethanaminylmethylene-2' -deoxyuridine;
3 ', 5 ' -bis (4-chlorobenzoyl) -5- [ 2-imidazolyl-4- ] ethanaminoylmethylene-2 ' -deoxyuridine;
5- (2-guanidinoethanoylmethylene) -2' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- (2-aminoethylaminoacylmethylene) -2 ' -deoxyuridine;
5 ' - (4,4 ' -dimethoxytriphenylmethyl) -5- [2- (N, N-dicyanoethoxycarbonyl) -guanidinoethanoylmethylene ] -2 ' -deoxyuridine;
5 '- (4, 4' -dimethoxytriphenylmethyl) -5- [2- (N, N-dicyanoethoxycarbonyl) -guanidinoethanoylmethylene ] -2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylaminephosphonamide.
A compound of formula I according to any one of the present invention, or a salt thereof, selected from the compounds shown in table 1 below:
table 1: part of the Compounds of the invention
The second aspect of the present invention relates to a process for producing the compound of the first aspect or a salt thereof, which comprises the following three production processes;
the first reaction formula of the preparation method is as follows:
the preparation method I comprises the following steps:
(1) reacting 2' -deoxyuridine-5-carboxylate with alkylenediamine, ethyl trifluoroacetate and triethylamine in methanol to obtain a compound represented by formula I (a);
(2) reacting the compound shown in the formula I (a) with ammonia water to obtain a compound shown in a formula I (b);
(3) reacting the compound shown in the formula I (a) with DMTr-Cl in the presence of pyridine to obtain a compound shown in a formula I (c);
(4) reacting the compound shown in the formula I (c) with N, N-diisopropylamine-tetrazole salt and a phosphorylation reagent in the presence of dichloromethane to obtain a compound shown in a formula I (d);
the above formula I (a), formula I (b), formula I (c), formula I (d) wherein m, n are as defined in any one of the compounds of the first aspect or salts thereof.
The second preparation method has the following reaction formula:
the preparation method II comprises the following steps:
(1) reacting 2' -deoxyuridine-5-carboxylic acid with histamine to obtain a compound represented by formula I (e);
(2) reacting the compound shown in the formula I (e) with tert-butoxy carbonate to obtain a compound shown in a formula I (f);
(3) reacting the compound shown in the formula I (f) with DMTr-Cl in the presence of pyridine to obtain a compound shown in a formula I (g);
(4) reacting the compound shown in the formula I (g) with N, N-diisopropylamine-tetrazole salt and a phosphorylation reagent in the presence of dichloromethane to obtain a compound shown in a formula I (h);
the above formula I (e), formula I (f), formula I (g), formula I (h) wherein m and n are as defined in any one of the compounds of the first aspect or salts thereof.
The preparation method has the following reaction formula:
the preparation method III comprises the following steps:
(1) reacting the compound shown in the formula I (b) with DMTr-Cl in the presence of pyridine to obtain a compound shown in a formula I (i),
(2) reacting the compound shown in the formula I (i) with N, N-bis (cyanoethoxy) -2-methyl-2-thioisothiourea to obtain a compound shown in a formula I (j),
(3) reacting a compound shown in a formula I (j) with N, N-diisopropylamine-tetrazole salt and a phosphorylation reagent in the presence of dichloromethane to obtain a compound shown in a formula I (k);
the definition of m, n in the above formula I (i), (j), formula I (k) is as described in any one of the compounds of the first aspect or the salts thereof.
In a third aspect the invention relates to a compound of formula II or a salt thereof,
wherein,
i is 0, 1, 2 or 3;
r is selected from hydroxyl, sulfhydryl, amino, -O (CH)2)mR’、-S(CH2)mR’、-NH(CH2)mR’、-N[(CH2)mR’]2And- (CH)2)mR'; wherein R' is selected from hydroxyl, sulfydryl, amino, guanidino and C5-20Aryl radical, C3-20Heterocyclyl, ester and amide groups, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n is 1, 2, 3, 4, 5, 6, 7 or 8.
In one embodiment of the third aspect of the present invention, R is-NH (CH)2)mR ', wherein R' is selected from amino, guanidino and imidazolyl, and m is 2 or 3.
In one embodiment of the third aspect of the present invention, n is 1, 2, 3, 4, 5 or 6, preferably 1 or 2.
In a fourth aspect, the invention relates to a nucleic acid which is modified with a nucleotide residue formed from a compound of formula II or a salt thereof according to the third aspect of the invention.
In one embodiment of the fourth aspect of the invention, the nucleotide residue formed by the compound of formula II or salt thereof is:
wherein,
i is 0, 1, 2 or 3;
n and R are as defined for any compound of the third aspect of the invention or a salt thereof;
preferably, in any of the nucleotide residues described above, the hydrogen in the hydroxyl group attached to the P atom may be replaced by a metal ion or an ammonium ion.
In one embodiment of the fourth aspect of the present invention, the nucleic acid is a deoxyribozyme or an aptamer.
In one embodiment of the fourth aspect of the present invention, the dnazyme is a dnazyme catalyzing cleavage of mRNA.
In one embodiment of the fourth aspect of the present invention, the mRNA is an mRNA of vascular endothelial growth factor receptor 2.
In one embodiment of the fourth aspect of the invention, the aptamer is an aptamer to human erythropoietin.
In one embodiment of the fourth aspect of the present invention, the nucleic acid is a deoxyribozyme formed by modifying SEQ ID NO. 1 with a nucleotide residue formed by a compound of formula II or a salt thereof according to the third aspect of the present invention.
SEQ ID NO:1 represents 5 '-d (tgc tct cca GGC TAG CTA CAA CGA cct gca cct) -3', also known as 10-23 deoxyribozyme. This is shown as 5 '-d (NNN NNN NNN GGC TAG CTA CAA CGA NNN NNN NNN) -3', in which the N-stretch represents the recognition domain of the 10-23 deoxyribozyme designed as a complementary sequence based on the base of the target sequence, and the middle portion GGC TAG CTA CAA CGA represents the catalytic domain of the 10-23 deoxyribozyme.
In one embodiment of the fourth aspect of the present invention, the dnazyme is selected from the group consisting of nucleic acids:
5’-d(tgc tct cca GGC TAG C2A CAA CGA cct gca cct)-3’(SEQ ID NO:3)；
5’-d(tgc tct cca GGC 2AG C2A CAA CGA cct gca cct)-3’(SEQ ID NO:4)；
5’-d(tgc tct cca GGC TAG C3A CAA CGA cct gca cct)-3’(SEQ ID NO:5)；
5’-d(tgc tct cca GGC 3AG C3A CAA CGA cct gca cct)-3’(SEQ ID NO:6)；
5’-d(tgc tct cca GGC 1AG C1A CAA CGA cct gca cct)-3’(SEQ ID NO:7)；
5’-d(tgc tct cca GGC TAG C4A CAA CGA cct gca cct)-3’(SEQ ID NO:8)；
5’-d(tgc tct cca GGC 4AG C4A CAA CGA cct gca cct)-3’(SEQ ID NO:9)。
in one embodiment of the fourth aspect of the invention, the nucleic acid is an aptamer formed by modifying SEQ ID NO. 2 with a nucleotide residue formed from a compound of formula II or a salt thereof according to the third aspect of the invention.
SEQ ID NO 2 stands for AAG GTC TGT TTT TGG GGT TGG TTT GGG.
In one embodiment of the fourth aspect of the present invention, the aptamer is selected from the group consisting of:
4AG GTC TGT TTT TGG GGT TGG TTT GGG(SEQ ID NO:10)；
A4G GTC TGT TTT TGG GGT TGG TTT GGG(SEQ ID NO:11)；
AAG GTC TGT TTT TGG GGT TGG 4TT GGG(SEQ ID NO:12)。
in one embodiment of the fourth aspect of the present invention, wherein the nucleotide residue represented by 1 is:
in one embodiment of the fourth aspect of the present invention, wherein the nucleotide residues represented by 2 are:
in one embodiment of the fourth aspect of the present invention, wherein the nucleotide residues represented by 3 are:
in one embodiment of the fourth aspect of the present invention, wherein the nucleotide residue represented by 4 is:
in one embodiment of the fourth aspect of the present invention, 1 to 4 representIn any nucleotide residue, the hydrogen in the hydroxyl group bonded to the P atom may be replaced with a metal ion or an ammonium ion (NH)4 +)。
A fifth aspect of the present invention relates to the use of a compound according to any one of the first aspect of the present invention or a salt thereof, or a compound according to any one of the third aspect of the present invention or a salt thereof, for the preparation of a nucleic acid.
In one embodiment of the fifth aspect of the present invention, the nucleic acid is a deoxyribozyme or an aptamer.
In one embodiment of the fifth aspect of the present invention, the dnazyme is a dnazyme catalyzing cleavage of mRNA.
In one embodiment of the fifth aspect of the invention, the mRNA is an mRNA of vascular endothelial growth factor receptor 2.
In one embodiment of the fifth aspect of the invention, the aptamer is an aptamer to human erythropoietin.
A sixth aspect of the invention relates to a composition or kit comprising a compound according to any one of the first aspect of the invention or a salt thereof, a compound according to claim 10 of the third aspect of the invention or a salt thereof, or a nucleic acid according to any one of the fourth aspect of the invention.
The seventh aspect of the present invention relates to a chip comprising the nucleic acid according to any one of the fourth aspects of the present invention.
The eighth aspect of the present invention relates to the use of the nucleic acid according to any of the fourth aspect of the present invention, the composition or the kit according to any of the sixth aspect of the present invention, or the chip according to any of the seventh aspect of the present invention for the preparation of a medicament for inhibiting vascular endothelial growth or for the detection of human erythropoietin.
A ninth aspect of the present invention relates to a method for treating vascular endothelial growth, which comprises administering to a subject in need thereof an effective amount of a dnazyme according to any one of the fourth aspects of the present invention.
The tenth aspect of the present invention relates to a method for detecting human erythropoietin, comprising the steps of:
(1) providing a sample to be tested;
(2) contacting an aptamer according to any one of SEQ ID NOs 10 to 12 of the fourth aspect of the invention with a sample to be tested;
(3) detecting the binding between the aptamer and human erythropoietin.
An eleventh aspect of the present invention relates to a compound according to any one of the first aspect of the present invention or a salt thereof, or a compound according to any one of the third aspect of the present invention or a salt thereof, which is used for producing a nucleic acid.
The twelfth aspect of the present invention relates to the nucleic acid according to any one of the fourth aspect of the present invention, the composition or the kit according to any one of the sixth aspect of the present invention, or the chip according to any one of the seventh aspect of the present invention, which is used for inhibiting vascular endothelial growth or detecting human erythropoietin.
In the present invention, terms are defined as follows:
the term "mercapto" refers to the abbreviation for sulfhydryl (-SH), also known as "thiol". A monovalent group consisting of two elements, hydrogen and sulfur.
The term "amino" is a monovalent radical of ammonia molecule removed from a hydrogen atom, as-NH2And (4) showing.
The term "C1-8Alkyl "refers to straight or branched chain alkyl groups having 1 to 8 carbon atoms. For example, a straight-chain or branched alkyl group having 1 to 6 carbon atoms, for example, a straight-chain or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-ethyl-butyl, hexyl, heptyl and octyl.
The term "C1-8Alkoxy "means" C1-8alkyl-O- ", wherein C1-8Alkyl is as defined above.
The term "benzyloxy" refers to the monovalent group remaining after removal of the hydrogen atom from the hydroxyl group in the benzyl alcohol (benzyl alcohol) molecule, and has the formula PhCH2O-。
The basic structure of the term "guanidino" is derived from guanidine, which has the formula:
the term "C5-20Aryl "refers to aromatic rings (including fused rings) containing 5 to 20 carbon atoms formally with one hydrogen atom eliminated, e.g. C6-20Aryl radical, C6-18Aryl radical, C6-12And (4) an aryl group. Examples of aryl groups specifically include cyclopentadienyl, phenyl, naphthyl, anthracenyl, fluorenyl and the like.
The term "C3-20The "heterocyclic group" means a heterocyclic group containing 3 to 20 carbon atoms and 1 to 3 hetero atoms selected from oxygen, nitrogen and sulfur, and is classified into two groups of an aliphatic heterocyclic group and an aromatic heterocyclic group. Examples of heterocyclyl groups include tetrahydrofuranyl, pyrrolidinyl, piperidinyl, morpholinyl, thiazolidinyl, thiazolinyl, thiathiazolinyl, imidazolyl, indolyl, benzimidazolyl, benzothiazolyl, thienyl, thiadiazolyl and the like.
The term "C3-20By "arylheterocyclyl" is meant a relatively stable ring system containing 3 to 20 carbon atoms, in which the ring, including the heteroatom, is planar and in which there are 4n +2 pi electrons in a ring-closing conjugated system, e.g. C3-12Aromatic heterocyclic group, C3-10Aromatic heterocyclic groups such as imidazolyl, indolyl, benzimidazolyl, benzothiazolyl and the like.
The term "ester group" has the formula-C (O) OR ", wherein R" is selected from C1-8Alkyl, hydroxy, mercapto, amino, guanidino, C5-20Aryl radical, C3-20Heterocyclic group, ester group, amide group, imidazole group; wherein, C1-8Alkyl radical, C5-20Aryl radical, C3-20The definition of heterocyclyl is as defined above.
The term "amido" refers to an amide (RC (O) NH2R') the monovalent radical remaining after removal of R in the molecule having the formula-C (O) NH2R '", wherein R'" is selected from C1-8Alkyl, hydroxy, mercapto, amino, guanidino, C5-20Aryl radical, C3-20Heterocyclic group, ester group, amide group, imidazole group; wherein, C1-8Alkyl radical, C5-20Aryl radical, C3-20The definition of heterocyclyl is as defined above.
The term "halogen" refers to group VIIA elements, including fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At).
The term "azido" has the formula-N3。
The term "O-allyl" has the formula-O-CH2-CH＝CH2。
The term "C1-8Alkoxy ethoxy "means" C1-8Alkoxy- (CH)2)2-O- ", wherein C1-8Alkoxy is as defined above.
The term "C1-8Alkylamino "refers to" C1-8alkyl-NH- ", wherein C1-8Alkyl is as defined above.
The term "C3-15Cycloalkylamino "means" C3-15cycloalkyl-NH- ". Wherein, C3-15Cycloalkyl represents a cyclic alkyl group having 3 to 15 carbon atoms. E.g. C3-8Cycloalkyl radical, C3-6Cycloalkyl radical, C3-5Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
The term "4-methoxytriphenylmethyl" has the formula:
the term "4, 4' -dimethoxytriphenylmethyl" has the structural formula:
the term "acetyl" refers to the residue of an acetic acid molecule after removal of the hydroxyl group, and has the formula CH3C(O)-。
The term "benzoyl" refers to the residue of a benzamide molecule after the amino group has been removed and has the formula PhC (O) -.
The term "tert-butyldimethylsilyl" (TBS) has the formula:
the term "tert-butyldiphenylsilyl" has the formula:
the term "trifluoroacetyl" refers to the residue of trifluoroacetamide molecule after removal of the amino group and has the formula CF3C(O)-。
The term "phthaloyl" refers to the divalent radical remaining after removal of two hydroxyl groups from a phthalic acid molecule and has the formula:
the term "cyanoethoxycarbonyl" has the formula:
the term "antisense nucleic acid" refers to a class of nucleic acid research techniques designed according to the principles of nucleic acid hybridization to selectively inhibit the expression of a particular gene, including three major techniques, antisense RNA, antisense DNA, and ribozymes.
The term "siRNA" refers to small interfering RNA, sometimes referred to as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), which is a double-stranded RNA 20 to 25 nucleotides in length that has many different biological uses.
The term "human erythropoietin" refers to a hormone-like substance secreted by the human kidney and liver and capable of promoting erythropoiesis.
The invention has the following beneficial effects:
1. the invention obtains a novel nucleoside compound or salt thereof.
2. The nucleic acid of the invention can be used as deoxyribozyme for catalyzing the mRNA cleavage of vascular endothelial growth factor receptor 2 or an aptamer of affinity human erythropoietin.
Detailed Description
Route 1:
(i)NH2(CH2)2NH2,in MeOH,at 50℃,CF3COOCH2CH3,EtN3,in methanol,r.t.；
(ii)conc.aq.ammonia,r.t.；
(iii)DMTr-Cl,in pyridine,at r.t.；
(iv)(NCCH2CH2O)[(iPr)2N]2P,(iPr)2EtN tetrazolium,in CH2Cl2,at r.t.
example 1: 5- [ N- (2-trifluoroacetamidoethylene) amidomethylene]-2' -deoxyuridine (compound)
1a) Synthesis of (2)
According to the scheme in scheme 1, 1.14g of 5-methoxycarbonylmethylene-2' -deoxyuridine (dU-EM, 3.8mol) was dissolved in 5mL of methanol, slowly dropped into 2.5mL of ethylenediamine (38mmol) in methanol, stirred for 4 hours, and when the reaction was complete by Thin Layer Chromatography (TLC), the solvent and the remaining ethylenediamine were evaporated under reduced pressure to give an oil. Dissolving the oily substance in 10mL of methanol, adding 2.1mL of triethylamine and 5mL of ethyl trifluoroacetate, mixing, fully mixing the mixture with silica gel, evaporating the solvent under reduced pressure, and separating by column chromatography to obtain 1.1g of a product (compound 1a) with the yield of 68.3 percent and Rf(dichloromethane: methanol, 9:1) 0.32.
1H NMR(400MHz,DMSO-d6):2.08(m,2H,C2’-H),3.05(s,2H,CH2),3.18(m,4H,CH2CH2),3.56(m,2H,C5’-H),3.77(m,1H,C4’-H),4.22(m,1H,C3’-H),4.96(t,J＝5.5,C5’-OH),5.24(d,J＝4.2,C3’-OH),6.17(t,J＝4.8,C1’-H),7.72(s,1H,C6-H),7.96(m,1H,CONH),9.37(s,1H,CONHCOCF3),11.34(s,1H,3-NH).
13C NMR(400MHz,DMSO-d6):35.3,39.5,63.4,72,4,85.9,89.3,110.5,116.4,119.3,140.4,152.2,158.2,158.6,165.2,171.7.
HRMS(C15H19F3N4O7+H+,425.1279):425.1278；(C15H19F3N4O7+Na+,447.1098):447.1097.
Example 2: synthesis of 5- (2-aminoethyl) -aminoacylmethylene-2' -deoxyuridine (Compound 1)
According to the scheme in scheme 1, compound 1a (400mg, 0.94mmol) was added to concentrated ammonia water (40mL), stirred at room temperature for 4 h and TLC showed reaction completion. Column chromatography to obtain 286mg of product, yield 93%, Rf(dichloromethane/ammonia methanol ═ 1:1) 0.47.
1H NMR(400MHz,DMSO-d6):2.09(m,2H,C2’-H),2.85(m,2H,CH2),3.10(s,2H,CH2),3.27(m,2H,CH2),3.53(m,2H,C5’-H),3.78(m,1H,C4’-H),4.24(m,1H,C3’-H),5.31(br,1H,C3’-OH),6.18(m,1H,C1’-H),7.77(s,1H,C6-H),8.09(m,1H,NH),8.55(br,1H,NH).
13C NMR(100MHz,DMSO-d6):33.9,37.1,61.9,70.9,84.5,87.9,109.0,139.0,150.9,163.8,170.8.
HRMS(C13H20N4O6+H+,329.1456):329.1456；(C13H20N4O6+Na+,351.1275):351.1276.
Example 3: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamido ethylidene) amide
Benzylidene radical]Synthesis of (E) -2' -deoxyuridine (Compound 1b)
According to the reaction scheme in scheme 1, 1.20g of Compound 1(2.82mmol) is dissolved in 5mL of dry pyridine, the solvent is evaporated under reduced pressure, and the above operation is repeated one more time to remove the water content of the starting material. The starting material was dissolved in 2mL of dry pyridine, to which 1.16g of DMTr-Cl (3.38mmol) was added in portions and reacted at room temperatureAfter 2 hours TLC indicated reaction was complete, column chromatography (5% pyridine on silica) gave 1.28g (Compound 1b) of product in 62.5% yield, Rf(dichloromethane: methanol, 9:1) 0.56.
1H NMR(400MHz,DMSO-d6):2.18(m,2H,C2’-H),2.70(s,2H,CH2),3.17(m,6H,CH2CH2,C5’-H),3.73(s,6H,2OCH3),3.87(m,1H,C4’-H),411.25(m,1H,C3’-H),5.32(d,J＝4.5,C3’-OH),6.20(t,J＝4.8,C1’-H),6.86,7.19-7.40(2m,13H,arom.H),7.55(s,1H,C6-H),7.86(m,1H,CONH),9.33(s,1H,CONHCOCF3),11.38(s,1H,3-NH).
13C NMR(400MHz,DMSO-d6):33.1,37.5,55.0,63.9,70.5,84.0,85.4,85.8,108.8,113.2,114.5,117.3,123.9,126.8,127.7,127.9,129.7,135.3,135.5,136.2,138.3,144.8,149.6,150.4,158.1,163.2,169.4.
HRMS(C36H37F3N4O9+Na+,749.2405):749.2405.
Example 4: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetamido ethylidene) amide
Benzylidene radical]Synthesis of (E) -2 '-deoxyuridine-3' - (2-cyanoethylene oxy) -N, N-diisopropylaminephosphonamide (Compound 1c)
Become into
According to the reaction scheme in scheme 1, 0.5g of compound 1b (0.688mmol) is dissolved in 10mL of redistilled dichloromethane, 0.14g N, N-diisopropylamine-tetrazolium salt and 0.10mL of phosphorylating reagent are added, the mixture is stirred at room temperature for 30 minutes, when TLC detection shows that the reaction is complete, the reaction solution is diluted with redistilled dichloromethane, washed once with cold 2% sodium bicarbonate solution, washed three times with saturated sodium chloride solution, dried with anhydrous sodium sulfate for 2 hours, filtered, concentrated, and subjected to column chromatography (5% pyridine) to obtain 0.42g of compound 1c, yield is 65.8%, R is separatedf(dichloromethane: methanol, 20:1) 0.42.
1H NMR(400MHz,CDCl3):1.16(m,12H,4CH3),2.36-2.76(m,6H,C2’-H,CH2,OCH2CH2CN),3.24-3.90(m,16H,NCH2CH2N,C5’-H,2OCH3,2CH,OCH2CH2CN),4.14(m,1H,C4’-H),4.70(m,1H,C3’-H),6.31(m,C1’-H),6.49(1H,NH),6.83,7.19-7.43(2m,13H,arom.H,C6-H),7.81(m,1H,CONH),7.89(s,1H,CONHCOCF3).
13C NMR(100MHz,DMSO-d6):20.2,22.2,23.2,24.6,34.7,38.9,40.5,43.4,55.3,62.4,62.6,85.2,87.0,108.8,112.7,113.4,113.6,117.7,127.5,128.1,128.3,128.5,130.2,135.2,138.8,144.2,150.0,158.2,158.8,170.0,171.3.
31P NMR:149.46,149.64.
HRMS(C45H54F3N6O10P+Na+,949.3483):949.3483.
Route 2:
(i)NH2(CH2)3NH2,in MeOH,at 50℃,CF3COOCH2CH3,EtN3,in methanol,r.t.；
(ii)conc.aq.ammonia,r.t.；
(iii)DMTr-Cl,in pyridine,at r.t.；
(iv)(NCCH2CH2O)[(iPr)2N]2P,(iPr)2EtN tetrazolium,in CH2Cl2,at r.t.
example 5: 5- [ N- (3-trifluoroacetamidopropylene) amidooxymethylene group]-2' -deoxyuridine (compound)
2a) Synthesis of (2)
According to the scheme in scheme 2, 3.42g of 5-methoxycarbonylmethylene-2' -deoxyuridine (dU-EM, 11.4mmol) was dissolved in 15mL of methanol, slowly dropped into 7.5mL of 114mmol propylenediamine-containing methanol in a 60 ℃ oil bath, stirred for 4 hours, and TLC showed that the reaction was complete, the solvent and the remaining propylenediamine were evaporated under reduced pressure to give an oil. Into oilAdding 6.3mL of triethylamine and 15mL of ethyl trifluoroacetate, and separating by column chromatography to obtain 3.30g (compound 2a) of a product with a yield of 66.1 percent, wherein R isf(dichloromethane: methanol, 9:1) 0.34.
1H NMR(400MHz,DMSO-d6):(ppm)1.60(m,2H,CH2CH2CH2),2.08(m,2H,C2’-H),2.98-3.10(m,6H,CH2,CH2CH2CH2),3.55(m,2H,C5’-H),3.77(m,1H,C4’-H),4.22(m,1H,C3’-H),5.00(br,1H,C5’-OH),5.27(br,1H,C3’-OH),6.16(t,J＝5.7Hz,1H,C1’-H),7.74(s,1H,C6-H),7.88(t,J＝5.3Hz,NH),9.40(s,1H,NH),11.34(s,1H,3-NH).
13C NMR(200MHz,DMSO-d6):(ppm)29.4,34.4,37.3,38.0,62.4,71.4,84.9,88.3,109.7,139.3,151.4,157.0,157.3,164.2,170.4.
HRMS(C16H21F3N4O7+H+,439.1435):439.1435；(C16H21F3N4O7+Na+,461.1255):461.1252.
Example 6: synthesis of 5- (3-aminopropylamidomethylene) -2' -deoxyuridine (Compound 2)
According to the scheme in scheme 2, compound 2a (500mg,1.14mmol) was added to concentrated ammonia water (45mL), stirred at room temperature for 4 h and TLC showed reaction completion. The product is obtained by column chromatography of 347mg with a yield of 88.9%, Rf(dichloromethane/ammonia methanol ═ 1:1) 0.51.
1H NMR(400MHz,DMSO-d6):(ppm)1.42(m,2H,CH2),2.05(m,2H,C2’-H),2.48(m,2H,CH2),3.05(m,4H,2CH2),3.53(m,2H,C5’-H),3.74(m,1H,C4’-H),4.20(m,1H,C3’-H),5.08(br,2H,NH2),6.15(t,J＝6.9,1H,C1’-H),7.70(s,1H,C6-H),7.81(m,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)33.8,34.3,37.3,62.3,71.3,84.9,88.3,109.7,139.1,151.4,164.2,170.1.
HRMS(C14H22N4O6+H+,343.1612):343.1611；(C13H20N4O6+Na+,365.1432):365.1429.
Example 7: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) amide
Benzylidene radical]Synthesis of (E) -2' -deoxyuridine (Compound 2b)
According to the reaction scheme in scheme 2, 1g of compound 2a (2.28mmol) is dissolved in 5mL of dry pyridine, the solvent is evaporated under reduced pressure, and the above operation is repeated again to remove the water content of the starting material. The starting material was dissolved in 2mL of dry pyridine, 0.94g of DMTrCl (2.74mmol) was added in portions and reacted at room temperature for 2 hours, and when TLC showed completion of the reaction, the product was isolated by column chromatography (5% pyridine) to give 1.04g (Compound 2b) in 61.6% yield, Rf(dichloromethane: methanol, 9:1) 0.56.
1H NMR(400MHz,DMSO-d6):(ppm)1.57(m,2H,CH2CH2CH2),2.19(m,2H,C2’-H),2.65(m,2H,C5’-H),2.98-3.26(m,6H,CH2,CH2CH2CH2),3.73(s,6H,2OCH3),3.88(m,1H,C4’-H),4.29(m,1H,C3’-H),5.36(d,J＝4.5Hz,1H,C3’-OH),6.22(t,J＝6.7Hz,1H,C1’-H),6.87(m,4H,arom.H),7.19-7.38(m,9H,arom.H),7.58(s,1H,C6-H),7.77(t,J＝5.6Hz,NH),9.39(s,1H,NH),11.41(s,1H,3-NH).
13C NMR(200MHz,DMSO-d6):(ppm)29.4,34.2,37.2,38.0,56.0,64.8,71.5,84.9,86.5,86.8,110.0,114.2,127.7,128.7,128.9,130.7,136.3,136.4,139.1,145.7,151.4,159.1,164.1,170.1.
HRMS(C37H39F3N4O9+Na+,763.2561):763.2561.
Example 8: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) aminyl
Methylene group]Synthesis of (E) -2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylaminephosphonamide (Compound 2c)
According to the reaction scheme in scheme 2, 0.5g of compound 2b (0.675mmol) is dissolved in 10mL of redistilled dichloromethane and 0.13g N, N-diisopropylamine-tetrazolium salt and 0.28 are added sequentiallymL of phosphorylating reagent, stirring for 30 minutes at room temperature, when TLC detection shows that the reaction is complete, redistilling dichloromethane to dilute the reaction solution, washing with cold 2% sodium bicarbonate solution once, washing with saturated sodium chloride solution three times, drying with anhydrous sodium sulfate for two hours, suction-filtering, concentrating the filtrate, and separating by column chromatography (5% pyridine) to obtain 0.43g of a product (compound 2c) with a yield of 67.7%, wherein R isf(dichloromethane: methanol, 20:1) 0.46.
1H NMR(400MHz,CDCl3):(ppm)1.14(m,12H),1.60(m,2H,CH2CH2CH2),2.34-2.68(6H,2CH,CH2CN,C2’-H),3.12-3.90(m,16H,CH2CH2CH2,C5’-H,CH2,OCH2,2OCH3),4.08(m,1H,C4’-H),4.69(m,1H,C3’-H),6.35(m,2H,C1’-H,NH),6.77-7.40(m,13H,arom.H),7.81(m,1H,C6-H),8.16(m,1H,NH),9.85(br,1H,NH).
13C NMR(200MHz,CDCl3):(ppm)20.5,20.6,20.7,23.2,23.3,24.7,24.8,24.9,25.0,29.2,36.2,36.3,36.4,43.4,43.5,43.6,55.5,55.6,58.3,58.5,63.0,63.1,73.2,73.4,85.2,85.3,85.5,86.0,87.2,109.4,113.6,117.8,118.1,127.5,128.4,128.6,130.5,135.5,135.6,139.2,144.4,150.5,157.5,159.0,164.4,164.5,171.1,171.2.
31P NMR(200MHz,CDCl3):(ppm)149.41.
HRMS(C46H56F3N6O10P+Na+,963.3640):963.3642.
Route 3:
(i)NH2(CH2)2NH2,in MeOH,at 50℃,CF3COOCH2CH3,EtN3,in methanol,r.t.；
(ii)conc.aq.ammonia,r.t.；
(iii)DMTrCl,in pyridine,at r..t.；
(iv)(NCCH2CH2O)[(iPr)2N]2P,(iPr)2EtN tetrazolium,in CH2Cl2,at r.t.
example 9: 5- [ N- (2-trifluoroacetamidoethylene) amidoethylene]-2' -deoxyuridine (compound)
3a) Synthesis of (2)
According to the scheme in scheme 3, 1.14g of 5-methoxycarbonylethylene-2' -deoxyuridine (dU-PM, 3.8mmol) was dissolved in 5mL of methanol, slowly dropped into 2.5mL of a 38mmol solution of ethylenediamine in an oil bath at 60 ℃ and stirred for 4 hours, and when TLC showed completion of the reaction, the solvent and the remaining ethylenediamine were evaporated under reduced pressure to give an oil. The oil was dissolved in 10mL of methanol, 2.1mL of triethylamine and 5mL of ethyl trifluoroacetate were added, the progress of the reaction was indicated by TLC, the reaction mixture was mixed with silica gel, the solvent was evaporated under reduced pressure, and column chromatography was performed to give 1.1g of the product (Compound 3a) in 68.3% yield, Rf(dichloromethane: methanol, 9:1) 0.34.
1H NMR(400MHz,DMSO-d6):2.08(m,2H,CH2),2.26(m,2H,C2’-H),2.43(m,2H,CH2),3.21(m,4H,CH2CH2),3.58(m,2H,C5-H),3.78(m,1H,C4’-H),4.25(m,1H,C3’-H),5.03(t,1H,J＝5.1,C5’-OH),5.26(d,J＝4.2,C3’-OH),6.18(t,1H,J＝6.9,C1’-H),7.66(s,1H,C6-H),7.98(m,1H,NH),9.43(m,1H,NH),11.32(s,1H,NH).
13C NMR(200MHz,DMSO-d6):23.8,35.1,38.3,62.3,71.4,84.8,88.3,113.6,137.5,151.3,164.3,172.6.
HRMS(C16H21F3N4O7+H+,439.1435):439.1434；(C16H21F3N4O7+Na+,461.1255):461.1255.
Example 10: 5- [ 2-aminoethyl group]Synthesis of Aminoacylethylene-2' -deoxyuridine (Compound 3)
According to the reaction scheme in scheme 3, Compound 3a (500mg,1.14mmol) is added to concentrated aqueous ammonia (45mL) and stirred at room temperature for 4 hoursTLC showed the reaction was complete. Column chromatography to obtain 368mg product with 94.6% yield, Rf(dichloromethane/ammonia methanol ═ 1:1) 0.50.
1H NMR(400MHz,DMSO-d6):2.08(m,2H,CH2),2.29(m,2H,C2’-H),2.45(m,2H,CH2),2.84(m,2H,CH2),3.26(m,2H,CH2),3.58(m,2H,C5-H),3.78(m,1H,C4’-H),4.25(m,1H,C3’-H),5.30(br,1H,C3’-OH),6.18(t,J＝6.9,1H,C1’-H),7.67(s,1H,C6-H),8.04(br,2H,NH,NH).
13C NMR(400MHz,DMSO-d6):23.2,34.6,37.0,61.9,71.0,84.4,87.8,113.1,137.1,150.8,163.8,172.7.
HRMS(C14H22F3N4O6+H+,343.1612):343.1611；(C14H22F3N4O6+Na+,365.1432):365.1433.
Example 11: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetylaminoethylidene) aminoyl
Ethylene radical]Synthesis of (E) -2' -deoxyuridine (Compound 3b)
According to the reaction scheme in scheme 3, 1.20g of Compound 3(2.82mmol) is dissolved in 5mL of dry pyridine, the solvent is distilled off under reduced pressure, and the above operation is repeated one more time to remove water from the starting material. The starting material was dissolved in 2mL of dry pyridine, and 1.16g of DMTr-Cl (3.38mmol) was added in portions and reacted at room temperature for 2 hours, and when the reaction was complete by TLC, the reaction was terminated by adding methanol. The reaction mixture was concentrated and separated by column chromatography (5% pyridine treatment) to give 1.28g (compound 3b) of the product, 62.5% yield, Rf(dichloromethane: methanol, 9:1) 0.58.
1H NMR(400MHz,DMSO-d6):2.09-2.26(m,6H,C2’-H,CH2CH2),3.13-3.20(m,6H,C5’-H,CH2CH2),3.73(s,6H,2CH3O),3.86(m,1H,C4’-H),4.22(m,1H,C3’-H),5.32(d,1H,J＝4.8,C3’-OH),6.16(t,1H,J＝6.7,C1’-H),6.89,71.9-7.41(m,14H,C6-H,arom.H),7.89(m,1H,NH),9.40(m,1H,NH),11.38(m,1H,NH).
13C NMR(200MHz,DMSO-d6):23.8,38.3,55.9,64.9,71.4,84.9,86.3,86.6,113.8,114.2,127.7,128.6,128.8,130.7,136.4,136.5,137.5,145.8,151.2,157.2,157.5,159.1,164.2,172.5.
HRMS(C37H39F3N4O9+Na+,763.2561):763.2561.
Example 12: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (2-trifluoroacetylaminoethylidene) aminoyl
Ethylene radical]Combination of (E) -2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylaminephosphonamide (Compound 3c)
Become into
According to the reaction scheme in scheme 3, 0.51g of compound 3b (0.663mmol) is dissolved in 10mL of dichloromethane, then 0.14g N, N-diisopropylamine-tetrazolium salt and 0.10mL of phosphorylating reagent are sequentially added, stirring is carried out at room temperature for 30 minutes, when TLC detection shows that the reaction is complete, the reaction solution is diluted with redistilled dichloromethane, washed once with cold 2% sodium bicarbonate solution, washed three times with saturated sodium chloride solution, then dried with anhydrous sodium sulfate for 2 hours, suction filtration is carried out, the filtrate is concentrated, and column chromatography (5% pyridine) is carried out to obtain 0.42g of product (compound 3c), the yield is 67.32%, R is Rf(dichloromethane: methanol, 20:1) 0.48.
1H NMR(400MHz,CDCl3):(ppm)1.14(m,12H,4CH3),2.04-2.64(m,10H,C2’-H,CH2CH2,2CH,CH2CN),3.27-3.88(m,16H,CH2CH2,C5’-H,2CH3O,OCH2),4.11(m,1H,C4’-H),4.63(m,1H,C3’-H),6.37(m,2H,C1’-H,NH),6.83,7.21-7.41(m,14H,C6-H,arom.H),7.60(s,1H,NH),8.22(br,1H,NH),9.80(br,1H,NH).
13C NMR(200MHz,CDCl3):(ppm)20.7,23.3,24.7,24.8,24.9,35.6,38.8,40.3,41.5,43.4,43.5,55.6,58.3,58.5,63.3,73.8,73.9,85.0,85.2,85.6,87.0,113..6,113.9,117.6,118.1,127.5,128.3,128.5,130.4,135.6,137.5,144.6,150.6,159.0,164.5,174.2.
31P NMR(200MHz,CDCl3):(ppm)149.26,149.35.
HRMS(C46H56F3N6O10P+Na+,963.3640):963.3642.
Route 4:
(i)NH2(CH2)2NH2,in MeOH,at 50℃,CF3COOCH2CH3,EtN3,in methanol,r.t.；
(ii)conc.aq.ammonia,r.t.；
(iii)DMTrCl,in pyridine,at r.t.；
(iv)(NCCH2CH2O)[(iPr)2N]2P,(iPr)2EtN tetrazolium,in CH2Cl2,at r.t.
example 13: 5- [ N- (3-trifluoroacetamidopropylene) amidoethylenyl ] ene]-2' -deoxyuridine (compound)
4a) Synthesis of (2)
According to the reaction scheme in scheme 4, 2.28g dU-PM (7.6mmol) is dissolved in 10mL of methanol, slowly added dropwise to 5mL of 76mmol propylenediamine-containing methanol in a 60 ℃ oil bath, stirred for 4 hours, and when the reaction is complete by TLC, the solvent and the remaining propylenediamine are evaporated under reduced pressure to give an oil. The oily substance was dissolved in 10mL of methanol, followed by addition of 4.2mL of triethylamine and 10mL of ethyl trifluoroacetate, stirring at room temperature, and separation by column chromatography to give 2.1g of the product (Compound 4a) in 61.1% yield, Rf(dichloromethane: methanol, 9:1) 0.36.
1H NMR(400MHz,DMSO-d6):(ppm)1.59(m,2H,CH2CH2CH2),2.06(m,2H,C2’-H),2.24,2.42(2t,J＝7.42Hz,4H,CH2CH2),3.01-3.18(m,4H,CH2CH2CH2),3.56(m,2H,C5’-H),3.76(m,1H,C4’-H),4.22(m,1H,C3’-H),5.00(t,J＝5.4,Hz,1H,C5’-OH),5.22(d,J＝4.2Hz,1H,C3’-OH),6.15(t,J＝7.1Hz,1H,C1’-H),7.63(s,1H,C6-H),7.84(m,1H,NH),9.36(s,1H,NH),11.29(s,1H,3-NH).
13C NMR(200MHz,DMSO-d6):(ppm)22.9,28.5,34.1,36.1,37.1,61.4,70.5,83.9,87.4,112.6,136.6,150.4,163.3,171.4.
HRMS(C17H23F3N4O7.H+,453.1592)453.1592；(C17H23F3N4O7.Na+,475.1411)475.1411.
Example 14: 5- [ (3-aminopropyl) -amineoylethylene]Synthesis of (E) -2' -deoxyuridine (Compound 4)
According to the scheme in scheme 4, compound 4a (300mg, 0.66mmol) was added to concentrated ammonia water (30mL), stirred at room temperature for 4 h and TLC showed reaction completion. Column chromatography to obtain 229mg, 97% yield, Rf(dichloromethane/ammonia methanol ═ 1:1) 0.53.
1H NMR(400MHz,DMSO-d6):1.67(m,2H,CH2),2.08(m,2H,C2’-H),2.28(m,2H,CH2),2.42(m,2H,CH2),2.78(m,2H,CH2),3.11(m,2H,CH2),3.59(m,2H,C5’-H),3.78(m,1H,C4’-H),4.25(m,1H,C3’-H),5.34(br,1H,C5’-OH),6.17(t,J＝6.9,1H,C1’-H),7.67(s,1H,C6-H),8.04(m,1H,NH,NH).
13C NMR(200MHz,DMSO-d6):23.3,28.0,34.5,36.0,37.2,61.9,70.9,84.4,87.8,113.1,116.1,119.1,137.0,150.8,158.9,159.2,163.8,172.3.
HRMS(C15H24N4O6.H+,357.1769)357.1769；C15H24N4O6.Na+,379.1588)379.1582.
Example 15: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) aminyl
Ethylene radical]Synthesis of (E) -2' -deoxyuridine (Compound 4b)
According to the reaction scheme in scheme 4, 1g of compound 4a (2.21mmol) is dissolved in 5mL of dry pyridine, the solvent is evaporated under reduced pressure, and the above operation is repeated one more time to remove the water content of the starting material. The starting material was dissolved in 2mL of dry pyridine,0.84g of DMTrCl (2.65mmol) was added thereto in portions and reacted at room temperature for 2 hours, and when the reaction was completed by TLC, the reaction was terminated by adding methanol. The reaction mixture was concentrated and separated by column chromatography (5% pyridine treatment) to give 1.18g (compound 4b) of compound (R), yield 70.7%)f(dichloromethane: methanol, 9:1) 0.55.
1H NMR(400MHz,DMSO-d6):(ppm)1.57(m,2H,CH2CH2CH2),2.12-2.25(m,6H,C2’-H,CH2CH2),2.98-3.18(m,6H,C5’-H,CH2CH2CH2),3.73(s,6H,2OCH3),3.86(m,1H,C4’-H),4.22(m,1H,C3’-H),5.32(d,J＝4.5Hz,1H,C3’-OH),6.16(t,J＝6.8Hz,1H,C1’-H),6.88(m,4H,arom.H),7.19-7.41(m,10H,C6-H,arom.H),7.76(m,1H,NH),9.38(s,1H,NH),11.39(s,1H,3-NH).
13C NMR(200MHz,DMSO-d6):(ppm)23.8,29.4,35.1,36.9,38.0,55.9,64.8,71.3,84.9,86.3,86.6,113.8,114.1,127.6,128.6,128.8,130.6,136.3,136.4,137.3,145.7,151.2,159.0,164.1,172.1.
HRMS(C38H41F3N4O9+H+,754.2826)754.2826；(C38H41F3N4O9+Na+,777.2713)777.2713.
Example 16: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [ N- (3-trifluoroacetamidopropylidene) -amine
Acylethylene radical]-2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylaminephosphonamide (Compound 4c)
Synthesis of (2)
According to the reaction scheme in scheme 4, compound 4b (0.5g, 0.66mmol) is dissolved in redistilled dichloromethane (10mL), stirred at room temperature, and to this solution N, N-diisopropylamine-tetrazolium salt (0.14g), phosphorylating agent (0.17mL) is added. After 30 minutes of reaction, the reaction was complete as checked by TLC. The reaction solution was diluted with redistilled dichloromethane and washed once with 2% cold sodium bicarbonate solution and three times with saturated sodium chloride. Drying the dichloromethane layer with anhydrous sodium sulfate for two hours, filtering, collecting the filtrate, and concentratingAnd (4) shrinking. Column chromatography (5% pyridine treatment) gave 0.45g of product, 71% yield. Rf(dichloromethane: methanol, 20:1) 0.39.
1H NMR(400MHz,DMSO-d6):(ppm)1.02-1.51(m,14H,2NCH(CH3)2,CH2CH2CH2),2.04-2.77(m,8H,C2’-H,CH2CH2,OCH2CH2CN),3.12-3.86(m,14H,2NCH(CH3)2,CH2CH2CH2,2CH3O,OCH2CH2CN),4.18(m,2H,C5’-H),4.61(m,1H,C4’-H),5.76(m,1H,C3’-H),6.36(m,1H,C1’-H),6.84,7.29(2m,14H,NH,arom.H),8.16(s,1H,C6-H),9.12(br,1H,NH),11.15(br,1H,NH).
31P NMR(160MHz,CDCl3):(ppm)149.26,149.40.
13C NMR(100MHz,CDCl3):(ppm)23.6,24.124.9,29.4,36.0,40.4,43.8,55.7,58.4,63.7,85.2,87.1,113..7,127.6,128.5,130.6,136.1,137.4,145.0,150.4,159.2,173.7.
HRMS(C47H58F3N6O10P+Na+,977.3796):977.3795.
Route 5:
example 17: 3 ', 5' -bis (4-chlorobenzoyl) -5- [ 2-imidazolyl-4-]Ethanaminoylmethylene-2' -deoxy
Synthesis of uridine (Compound 5a)
According to the scheme in scheme 5, 3 ', 5 ' -bis (p-chlorobenzoyl) -2 ' -deoxyuridine-5-acetic acid (5.6g, 10mmol) is suspended in dichloromethane (50mL) and N, N-dimethylformamide (15mL) is added and mixed well with stirring to give a clear solution. DCC (2.25g, 10.09mmol) and HOSu (1.25g, 10.85mmol) were added to the solution. Stirring at room temperature overnight, detecting the reaction by TLC, completely reacting the raw materials, and simultaneously separating out white precipitate from the reaction solution. Histamine (1.34g, 12 mmol) was added to the reaction mixture) After stirring at room temperature for 4 hours, the reaction was completed. The precipitate was removed by suction filtration and an equal volume of 5% NaHCO was added to the filtrate3Extracting the solution, taking the lower organic phase, adding a proper amount of anhydrous sodium sulfate and drying. The filtrate was thoroughly mixed with silica gel and the solvent was removed under reduced pressure, and the product was isolated by column chromatography in 56.5% yield and Rf (dichloromethane: methanol, 9:1)0.13 of 3.7 g.
1H NMR(400MHz,DMSO-d6):(ppm)2.59(m,2H,C2’-H),2.78(m,2H,CH2),2.97(s,2H,CH2),3.24(m,2H,CH2),4.46-4.64(m,3H,C4’-H,C5’-H),5.63(m,1H,C3’-H),6.31(t,J＝7.2,1H,C1’-H),7.54-7.68,7.94-8.08(2m,10H,arom.H,2CH),8.98(s,1H,NH),11.46(s,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)25.3,34.1,38.7,39.9,65.5,75.9,81.9,85.7,110.2,117.0,129.1,130.0,132.0,132.2,132.3,134.5,139.5,139.6,151.3,164.1,165.5,165.7,170.3.
HRMS(C30H27Cl2N5O8+H+,656.1309)656.1308；(C30H27Cl2N5O8+Na+,678.1129)678.1125.
Example 18: 5- [ 2-imidazolyl-4-]Synthesis of ethanaminylmethylene-2' -deoxyuridine (Compound 5)
The method comprises the following steps: compound 5a (4g, 6.11mmol) was dissolved in sodium methoxide/methanol solution (0.1M, 180mL) and stirred at room temperature for 3 hours until the TLC detection reaction was complete. Subsequently, acetic acid was added to adjust the pH to neutral. Mixing the reaction solution with silica gel, and performing column chromatography to obtain product 2.27g with yield of 98%, Rf(dichloromethane: methanolic ammonia, 9:1) 0.05.
The second method comprises the following steps: compound 5a (4g, 6.11mmol) was dissolved in sodium methoxide/methanol solution (0.1M, 180mL) and stirred at room temperature for 3 hours until the TLC detection reaction was complete. Subsequently, acetic acid was added to adjust the pH to neutral. Evaporating the reaction solution under reduced pressure to obtain a little viscous liquid, slowly dripping the viscous liquid into rapidly stirred dichloromethane (500mL), stirring for 2 hours, standing, and performing suction filtration to obtain a white solid product 2.27g, wherein the yield is 98%, and R isf(Dichloromethane)Alkane, ammonia methanol, 9:1) 0.05.
1H NMR(400MHz,DMSO-d6):(ppm)2.09(m,2H,C2’-H),2.78(m,2H,CH2),3.06(s,2H,CH2),3.17(m,2H,CH2),3.56(m,2H,C5’-H),3.78(m,2H,C4’-H),4.25(m,1H,35’-H),5.10(br,2H,C3’-OH,C5’-OH),6.11(t,J＝6.8,1H,C1’-H),7.44(s,1H,CH),7.78(s,1H,C6-H),8.09(m,1H,NH),9.03(s,1H,CH),11.36(s,1H,NH),14.47(br,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)24.3,33.4,37.7,48.6,61.4,70.4,81.5,84.0,87.4,108.7,116.1,130.9,133.5,138.5,150.4,163.3,169.6.
HRMS(C16H21N5O6+H+,380.1565)380.1565；C43H46N8O12+Na+,402.1384)402.1381.
Route 6:
example 19: 5 ' - (4,4 ' -Dimethoxytriphenylmethyl) -5- (2-aminoethylaminoacylmethylene) -2 ' -deoxy
Synthesis of Oxuridine (Compound 6a)
According to the reaction scheme in scheme 6, compound 1b (1g, 1.38mmol) is added to concentrated ammonia water (40mL), sealed and stirred at room temperature for 3 hours, TLC shows complete reaction, equal volume of ether is added for extraction, water phase is taken after separation, and product 0.84g is obtained after spin-drying with yield 96.8%, Rf(dichloromethane/methanol ═ 9:1) 0.17.
1H NMR(400MHz,DMSO-d6):(ppm)2.17(m,2H,C2’-H),2.96,3.16(2m,4H,CH2CH2),3.70(s,6H,2OCH3),3.84(m,1H,C4’-H),4.24(m,1H,C3’-H),5.31(d,J＝4.5,1H,C3’-OH),6.18(t,J＝6.7,1H,C1’-H),6.85,7.16-7.36(2m,14H,arom.H,NH),7.53(s,1H,C6-H),7.66(m,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)33.8,41.7,42.7,55.6,64.4,71.0,84.4,86.0,86.3,109.7,113.8,127.3,128.2,128.5,130.2,135.8,136.0,138.6,145.3,150.9,158.6,163.7,169.5.
HRMS(C34H38N4O8+Na+,653.2582)653.2579.
Example 20: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [2- (N, N-dicyanoethoxycarbonyl) -guanidinoethylene
Aminoacylmethylene]Synthesis of (E) -2' -deoxyuridine (Compound 6b)
According to the reaction scheme in scheme 6, compound 6a (0.8g, 1.27mmol) was dissolved in N, N-dimethylformamide (4.4mL), guanidino (0.4g, 1.41mmol) and triethylamine (0.177mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 4 hours and TLC was used to check completion of the reaction. 5% NaHCO was added to the reaction solution in turn3Stirring the solution (20mL) and ethyl acetate (60mL) at normal temperature for 10 minutes, standing, taking an organic phase after the organic phase is separated, drying the organic phase by using anhydrous sodium sulfate, fully mixing the dried organic phase with silica gel, removing the solvent under reduced pressure, and separating by column chromatography to obtain 0.75g of a product, wherein the yield is 68.6 percent, and Rf (dichloromethane: methanol, 9:1) is 0.52.
1H NMR(400MHz,DMSO-d6):(ppm)2.20(m,2H,C2’-H),2.69(m,2H,CH2),2.88(t,J＝6.0,2H,OCH2CH2CN),2.98(t,J＝6.0,2H,OCH2CH2CN),3.14-3.28(m,NCH2CH2N),3.38(m,2H,C5’-H),3.74(s,6H,2OCH3),3.89(m,1H,C4’-H),4.18(t,J＝5.9,2H,OCH2CH2CN),4.28(m,1H,C3’-H),4.34(t,J＝5.9,2H,OCH2CH2CN),5.37(d,J＝4.5,1H,C3’-OH),6.23(t,J＝6.9,1H,C1’-H),6.89,7.12-7.40(2m,13H,arom.H),7.58(s,1H,C6-H),7.90(m,1H,NH),8.49(t,J＝5.6,1H,NH),11.40(s,1H,NH),11.54(s,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)18.2,18.3,33.9,38.7,55.6,55.8,60.8,61.9,64.6,71.2,71.3,84.8,86.3,86.6,109.7,114.0,119.2,119.7,127.6,128.4,128.7,130.5,136.1,136.3,138.9,145.5,151.2,152.7,156.1,158.9,163.3,163.9,170.2.
HRMS(C43H46N8O12+Na+,889.3127)889.3125.
Examples21: 5 '- (4, 4' -Dimethoxytriphenylmethyl) -5- [2- (N, N-dicyanoethoxycarbonyl) -guanidinoethylene
Aminoacylmethylene]-2 '-deoxyuridine-3' - (2-cyanoethylene-O-) -N, N-diisopropylaminephosphonamide (compound)
6c) Synthesis of (2)
According to the reaction scheme in scheme 6, compound 6b (0.4g, 0.45mmol) is dissolved in redistilled dichloromethane (10mL), stirred at room temperature, and to this solution N, N-diisopropylamine-tetrazolium salt (0.10g), phosphorylating agent (0.12mL) is added. After 30 minutes of reaction, TLC detection was carried out, and the reaction was complete. The reaction solution was diluted with redistilled dichloromethane and washed once with 2% cold sodium bicarbonate solution and three times with saturated sodium chloride. The dichloromethane layer was dried over anhydrous sodium sulfate for two hours, filtered under suction, and the filtrate was concentrated. Column chromatography (5% pyridine treatment) gave 0.42g of product, 87% yield. Rf (dichloromethane: methanol, 20:1) 0.41.
1H NMR(400MHz,CDCl3):(ppm)1.00-1.26(m,14H,2iPr),2.36(m,1H,C2’-H),2.50-2.76(m,9H,C2’-H,CH2,3OCH2CH2CN),3.20-3.56(m,6H,NCH2CH2N,C5’-H),3.68-3.88(m,8H,OCH2CH2CN,2OCH3),4.06(m,1H,C4’-H),4.23-4.38(m,4H,2OCH2CH2CN),4.67(m,1H,C3’-H),6.28(t,J＝6.4,1H,C1’-H),6.54(t,J＝5.8,1H,NH),6.83,7.18-7.40(2m,14H,arom.H,),7.71(m,1H,NH),7.74(s,1H,C6-H),8.33(t,J＝5.5,1H,NH),11.65(s,1H,NH),11.69(s,1H,NH).
13C NMR(100MHz,DMSO-d6):(ppm)18.4,18.5,20.7,20.8,24.8,35.2,39.0,40.6,41.2,43.5,43.6,55.6,58.3,58.4,60.1,61.0,62.9,73.0,73.2,85.2,87.1,109.4,113.6,116.8,117.5,118.3,127.5,128.4,128.5,130.5,135.7,138.8,144.5,150.2,153.0,156.6,159.0,163.2,163.8,170.4.
32P NMR(100MHz,DMSO-d6):(ppm)149.44.
HRMS C52H63N10O13P+Na+,1089.4206)1089.4203.
Example 22: synthesis of functional nucleic acid sequences
(1) Compounds 1-4 as nucleoside monomers, nucleoside monomer derivatives 1b, 1c, 2b, 2c, 3b, 3c, 4b or 4c were immobilized on CPG resin, and the DMTr group on nucleoside monomer 5, -OH was removed using dichloromethane containing 3% trichloroacetic acid to be in a free state.
(2) The phosphoramidite monomer is treated by tetrazole to protonate a diisopropylamine group on the phosphoramidite monomer into a good leaving molecule, then the treated phosphoramidite monomer and 5' -OH on the nucleoside monomer are subjected to condensation reaction, and the reaction time of the non-natural nucleoside monomer and the phosphoramidite monomer is prolonged to 300 seconds.
(3) In order to ensure the accuracy of sequence synthesis, 5 '-OH of a small amount of nucleoside monomers which cannot participate in the reaction on the resin is blocked, and the 5' -OH is acetylated by using acetic anhydride and 1-methylimidazole.
(4) The phosphoramidite on the reaction product is oxidized to a phosphotriester with iodine.
(5) Cutting nucleic acid sequence from resin with strong ammonia water, incubating to remove protecting groups on base and phosphate group, concentrating, purifying by gel electrophoresis, desalting, and identifying.
Test example 1: experiment for catalyzing cleavage of mRNA (VEGFR 2 mRNA) of vascular endothelial growth factor receptor 2
The 10-23 deoxyribozyme (DZ01) has catalytic cleavage effect on mRNA of vascular endothelial growth factor receptor 2 (VEGFR 2 mRNA). 10-23 deoxyribozymes were structurally modified with the compounds 1, 2, 3, and 4 of the present invention, and the modified deoxyribozymes were synthesized according to the method for synthesizing functional nucleic acid sequences in example 22, and their molecular weights are shown in Table 2.
The catalytic cleavage capacity of the deoxyribozymes to mRNA before and after modification is examined by the following method:
a fragment of mRNA whose substrate is derived from the vascular endothelial growth factor receptor 2, was used in the assay in the form of a DNA-RNA-DNA chimera, i.e., 5' -d (AGG TGC AGG) -rAU-d (TG GAG AGC A) -3. By using 5' - [ gamma-32P]ATP labelling, concentration of deoxyribozymes and substrates under multiple conversion conditionsThe ratio was 100:1, the reaction system contained 50mM Tris-HCl and 2mM MgCl2+The pH was maintained at 7.5. Samples were taken at various time points and analyzed by 20% denaturing gel electrophoresis, and the intensity of the radiation exposure was used as the concentration ratio of the reactants and products, and the apparent reaction rate constants were calculated according to the Hoofstee equation described below, and the results are shown in Table 3.
P＝P∞-C.exp[-kobst]
Wherein:
p represents the percentage (%) of the product at time point t;
P∞the percentage concentration (%) of the product at the end of the reaction;
c represents the difference (%) in the percentage concentration of the product between the end point and the starting point of the reaction;
kobsindicates the apparent reaction rate constant (min)-1)。
TABLE 2
TABLE 3
| Deoxyribozymes | Modification means (site) | kobs(min-1) |
| SEQ ID：1 | 0.0051±0.0005 | |
| SEQ ID：3 | T8＝2 | 0.0054±0.0008 |
| SEQ ID：4 | T4＝T8＝2 | 0.0052±0.0007 |
| SEQ ID：5 | T8＝3 | 0.0088±0.0005 |
| SEQ ID：6 | T4＝T8＝3 | 0.0064±0.0006 |
| SEQ ID：7 | T4＝T8＝1 | 0.0062±0.0006 |
| SEQ ID：8 | T8＝4 | 0.0072±0.0008 |
| SEQ ID：9 | T4＝T8＝4 | 0.0059±0.0005 |
As can be seen from tables 2-3, after 10-23 DNAzymes were modified with the compounds 1-4 of the present invention, the catalytic cleavage ability of the novel DNAzymes for the mRNA of VEGF receptor 2 was improved.
Test example 2: affinity assay for aptamers to human erythropoietin
Aptamer In27 is an aptamer to human erythropoietin. Aptamer In27 was structurally modified with compound 4 of the present invention, and a modified nucleic acid (aptamer) was synthesized by the nucleic acid sequence synthesis method In example 22, and the modified aptamer and its molecular weight are shown In table 4.
The binding constants of the aptamers to human erythropoietin before and after modification were measured by the SPR method, and the smaller the binding constant, the better the affinity was, and the results are shown in Table 5.
TABLE 4
TABLE 5
As is clear from tables 4 to 5, In27 was structurally modified with Compound 4 of the present invention, and the binding affinity between the obtained aptamer and human erythropoietin was significantly improved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
SEQUENCE LISTING
<110> institute of poison drug of military medical science institute of liberty military of China
<120> nucleoside compound or salt thereof, nucleic acid and use thereof
<130> IDC170074
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 33
<212> DNA
<213> deoxyribozyme
<400> 1
tgctctccag gctagctaca acgacctgca cct 33
<210> 2
<211> 27
<212> DNA
<213> aptamer
<400> 2
aaggtctgtt tttggggttg gtttggg 27
<210> 3
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 2
<400> 3
tgctctccag gctagcnaca acgacctgca cct 33
<210> 4
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (13)..(13)
<223> n is residue represented by formula 2
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 2
<400> 4
tgctctccag gcnagcnaca acgacctgca cct 33
<210> 5
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 3
<400> 5
tgctctccag gctagcnaca acgacctgca cct 33
<210> 6
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (13)..(13)
<223> n is residue represented by formula 3
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 3
<400> 6
tgctctccag gcnagcnaca acgacctgca cct 33
<210> 7
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (13)..(13)
<223> n is residue represented by formula 1
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 1
<400> 7
tgctctccag gcnagcnaca acgacctgca cct 33
<210> 8
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 4
<400> 8
tgctctccag gctagcnaca acgacctgca cct 33
<210> 9
<211> 33
<212> DNA
<213> deoxyribozyme
<220>
<221> misc_feature
<222> (13)..(13)
<223> n is residue represented by formula 4
<220>
<221> misc_feature
<222> (17)..(17)
<223> n is residue represented by formula 4
<400> 9
tgctctccag gcnagcnaca acgacctgca cct 33
<210> 10
<211> 27
<212> DNA
<213> aptamer
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is residue represented by formula 4
<400> 10
naggtctgtt tttggggttg gtttggg 27
<210> 11
<211> 27
<212> DNA
<213> aptamer
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is residue represented by formula 4
<400> 11
anggtctgtt tttggggttg gtttggg 27
<210> 12
<211> 27
<212> DNA
<213> aptamer
<220>
<221> misc_feature
<222> (22)..(22)
<223> n is residue represented by formula 4
<400> 12
aaggtctgtt tttggggttg gnttggg 27
Claims (24)
1. A compound of formula I or a salt thereof,
wherein,
r is-NH (CH)2)mR'; wherein R' is substituted or unsubstitutedAmino, m is 2 or 3;
R1is-H;
R2is-H or 4, 4' -dimethoxytriphenylmethyl;
R3is-H or PN (iPr)2OCH2CH2CN；
n is 1 or 2;
wherein the substituted amino group is an amino group which is mono-or polysubstituted with the following substituents: trifluoroacetyl group.
2. The compound or salt thereof according to claim 1, wherein R' is amino.
3. The compound or salt thereof according to claim 1, wherein R' is a substituted amino group.
4. The compound or salt thereof according to claim 1, which is selected from the following compounds and salts thereof:
5. a compound of formula II or a salt thereof,
wherein,
i is 0, 1, 2 or 3;
r is-NH (CH)2)mR'; wherein R' is amino, m is 2 or 3;
n is 1 or 2.
6. A nucleic acid which is a nucleic acid resulting from a modification of a nucleotide residue formed by a compound of formula ii of claim 5 or a salt thereof.
7. The nucleic acid of claim 6, which is a deoxyribozyme or an aptamer.
8. The nucleic acid according to claim 7, wherein the dnazyme is a dnazyme catalyzing cleavage of mRNA.
9. The nucleic acid of claim 8, wherein the mRNA is a vascular endothelial growth factor receptor 2 mRNA.
10. The nucleic acid of claim 7, wherein the aptamer is an aptamer to human erythropoietin.
11. The nucleic acid according to any one of claims 6 to 10, which is a deoxyribozyme formed by modification of SEQ ID No. 1 with nucleotide residues formed by a compound of formula ii according to claim 5 or a salt thereof.
12. The nucleic acid of claim 11, wherein the dnazyme is selected from the group consisting of:
5’-d(tgc tct cca GGC TAG C2A CAA CGA cct gca cct)-3’(SEQ ID NO:3)；
5’-d(tgc tct cca GGC 2AG C2A CAA CGA cct gca cct)-3’(SEQ ID NO:4)；
5’-d(tgc tct cca GGC TAG C3A CAA CGA cct gca cct)-3’(SEQ ID NO:5)；
5’-d(tgc tct cca GGC 3AG C3A CAA CGA cct gca cct)-3’(SEQ ID NO:6)；
5’-d(tgc tct cca GGC 1AG C1A CAA CGA cct gca cct)-3’(SEQ ID NO:7)；
5’-d(tgc tct cca GGC TAG C4A CAA CGA cct gca cct)-3’(SEQ ID NO:8)；
5’-d(tgc tct cca GGC 4AG C4A CAA CGA cct gca cct)-3’(SEQ ID NO:9)；
wherein, the nucleotide residues represented by 1 are:
2 is represented by the following nucleotide residues:
3 are:
4 are:
13. the nucleic acid according to claim 12, wherein the hydrogen in the hydroxyl group bonded to the P atom in any one of the nucleotide residues represented by 1 to 4 is replaced with a metal ion or an ammonium ion.
14. The nucleic acid according to claim 6 or 7 or 10 which is an aptamer formed by modification of SEQ ID No. 2 with a nucleotide residue formed from a compound of formula ii according to claim 5 or a salt thereof.
15. The nucleic acid of claim 14, wherein the aptamer is selected from the group consisting of:
4AG GTC TGT TTT TGG GGT TGG TTT GGG(SEQ ID NO:10)；
A4G GTC TGT TTT TGG GGT TGG TTT GGG(SEQ ID NO:11)；
AAG GTC TGT TTT TGG GGT TGG 4TT GGG(SEQ ID NO:12)；
in SEQ ID NO 10, the nucleotide residues represented by 4 are:
11-12, nucleotide residues represented by 4 are:
16. the nucleic acid according to claim 15, wherein the hydrogen in the hydroxyl group bonded to the P atom in the nucleotide residue represented by 4 is replaced with a metal ion or an ammonium ion.
17. Use of a compound according to any one of claims 1 to 4 or a salt thereof or a compound according to claim 5 or a salt thereof for the preparation of a nucleic acid.
18. The use of claim 17, wherein the nucleic acid is a deoxyribozyme or an aptamer.
19. The use of claim 18, wherein the dnazyme is a dnazyme that catalyzes mRNA cleavage.
20. The use of claim 19, wherein the mRNA is an mRNA of vascular endothelial growth factor receptor 2.
21. The use of claim 18, wherein the aptamer is an aptamer to human erythropoietin.
22. A composition or kit comprising a compound or salt thereof of any one of claims 1 to 4, a compound or salt thereof of claim 5, or a nucleic acid of any one of claims 6 to 16.
23. A chip comprising the nucleic acid of any one of claims 6 to 16.
24. Use of the nucleic acid of any one of claims 6 to 16, the composition of claim 22, or the chip of claim 23 for the manufacture of a medicament for inhibiting vascular endothelial growth or for the manufacture of a kit for the detection of human erythropoietin.
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| CN1814614A (en) * | 2005-02-06 | 2006-08-09 | 中国人民解放军军事医学科学院毒物药物研究所 | Nucleic acid, peptide nucleicacid derivatives and their use |
| CN102766183A (en) * | 2011-05-05 | 2012-11-07 | 中国人民解放军军事医学科学院毒物药物研究所 | Anti-HIV-1 fusion activity-modified nucleic acid structure |
| US20150315541A1 (en) * | 2012-12-13 | 2015-11-05 | Moderna Therapeutics, Inc. | Modified polynucleotides for altering cell phenotype |
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| CN1814614A (en) * | 2005-02-06 | 2006-08-09 | 中国人民解放军军事医学科学院毒物药物研究所 | Nucleic acid, peptide nucleicacid derivatives and their use |
| CN102766183A (en) * | 2011-05-05 | 2012-11-07 | 中国人民解放军军事医学科学院毒物药物研究所 | Anti-HIV-1 fusion activity-modified nucleic acid structure |
| US20150315541A1 (en) * | 2012-12-13 | 2015-11-05 | Moderna Therapeutics, Inc. | Modified polynucleotides for altering cell phenotype |
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